Many methods are known for detecting biological trace components, which are highly sensitive and specific. Briefly, in those methods a targeted trace analyte is detected or quantitatively determined by labeling the analyte with a suitable labeling material, immobilizing the labeled analyte on a suitable medium based on a specific binding reaction, thoroughly washing it, and then detecting the labeling material by suitable means. Examples of the specific binding reactions generally used are the antigen-antibody reaction, the avidin-biotin binding reaction, and the receptor-ligand binding reaction.
Labeling materials and methods for label detection are known to be of various types depending on the chemical and physical properties of the labeling materials. Usually, such methods are as follows: (1) The sum of signals, e.g., radiation doses, amounts of fluorescence, or amounts of luminescence (chemiluminescence, bioluminescence), from the trace analyte labeled with a labeling material and immobilized on a suitable solid phase is measured, and presence of the trace analyte is evaluated, and quantitatively determined, based on the correlation between measured values and concentrations of the trace analyte. (2) The number of the labeled analyte molecules immobilized on a suitable solid phase is counted by observing phenomena such as radiation, fluorescence, or luminescence (chemiluminescence, bioluminescence) from the labeling material, and presence of the trace analyte is evaluated, and quantitatively determined, based on the count. Method (2) requires that the trace labeled analytes immobilized on the solid phase are counted molecule by molecule. Compared with method (1), method (2) may have advantages, such as lower background noise, shorter measuring time, and decreased measurement errors (increased measurement sensitivity).
Thus, it is desirable to develop a method which comprises counting, individually, trace labeled analytes fixed to a suitable solid phase, based on presence or absence of signals from the labeling material, in order to evaluate the presence of the analyte and quantitatively determine the analyte.
The usual, known method of labeling an analyte with a fluorescent molecule should give single molecule detection under idealized conditions. However, it is difficult to use this method for the above-mentioned ultrahigh sensitivity detection under practical measuring conditions, which involves use of an ordinary fluorescence microscope. The amount of fluorescence emitted from a single fluorophore is usually very small partly because of rapid bleaching of the fluorochrome. That is, such signals from the labeled analyte are very small and weak. Since background fluorescence is also comparable with signal fluorescence, measurement of individual luminescence phenomena is virtually unpractical.